Abstract: To identify the genetic background and relationship of Stylosanthes varieties introduced newly and promote the further utilization of them，the genetic diversity of 31 Stylosanthes varieties introduced newly and 4 cultivars was analyzed using 23 SSR markers. Results showed that all 23 SSR markers were polymorphic among 35 Stylosanthes materials，and a total of 134 alleles were generated by these SSR markers. The number of alleles of SSR markers ranged from 3 to 11 with an average of 5.83 alleles. The polymorphic information content each SSR marker varied from 0.515 to 0.845，with an average of 0.703. Based on the results of analysis of cluster and principal components，the 35 Stylosanthes materials were divided into 5 separate clusters，and the lower genetic relation was presented between cluster III and 4 cultivars，cluster V and 4 cultivars.